Method for producing a mammal provided with resistance to an alpha-herpes virus mediated infection and mamal obtained by implementing said method and said mamal&#39;s progeny

ABSTRACT

The invention concerns a method for producing a mammal belonging to a non-human species made resistant by germinal transgenesis to an alpha-herpes virus mediated infection, for which the HveC or nectin-1 polypeptide constitutes a functional receptor. The method consists in introducing by insertion or homologous recombination into the genome of the cells constituting the mammal&#39;s germinal line, a transgene enabling expression of a chimeric protein consisting of an extracellular domain of nectin-1 or HveC or one of its parts and an immunoglobulin crystallzable fragment, in a suitable expression system.

Different viruses of the herpes type which are distinguished by theirgenome and their biological characteristics are known.

A subfamily of these viruses corresponds to the alphaherpesvirus,examples of which include the human herpes simplex virus type 1 and type2 (HSV-1 and HSV-2), the Aujeszky's disease virus or Pseudorabies virus(PRV) and bovine herpes virus type 1 (BHV-1).

All these viruses have the characteristic of being neurotropes andhaving a very short replication cycle and a broad host spectrum.

Infection by these viruses causes lesions of the epidermis, situated asa rule in the mucous membranes, followed by spreading of the virus tothe nervous system which can involve acute inflammations and latentinfections.

Examples of the alphaherpesviruses involving the greatest damage are thePRV virus which is a pathogenic agent of major economic importance inpig production due to the direct cost of the pathologies caused and thusto the means for combating it employed.

This virus is widely present in the majority of regions of high pigproduction (Europe, North America and Asia).

There are currently several vaccines against the PRV virus whichrepresent a major market worldwide.

Such a vaccination is not however without disadvantages considering inparticular the cost involved in the need to vaccinate a large proportionof animals in a herd and practical problems linked with this which makethis operation particularly awkward, all the more so because in generalit is necessary to carry out several injections.

The cost and constraints of this operation can limit its use and thuseven also its effectiveness.

Strategies for eradicating the virus are also used with variable butalways weak results.

The BHV-1 virus responsible for infectious bovine rhinotracheitis againcauses major problems in farming.

This virus is in fact highly contagious both in new-born calves and inolder animals; it can cause inflammation of the nasal cavity and thelarynx, ocular lesions and respiratory problems, but can also occur inthe brain causing encephalitis or even take on genital forms.

These different problems which are frequently fatal in the new-born calfmean major losses for farmers; moreover, in dairy cattle a dramatic fallin milk production can be observed.

There are also several vaccines against the BHV-1 virus, but vaccinationhas the same disadvantages as vaccination against the PRV virus in pigfarming.

Furthermore, vaccines against the BHV-1 virus injected by intra-muscularadministration have been considered responsible for abortions inpregnant cows.

Eradication of the virus has also been proposed in particular in someEuropean countries, but it has proved particularly difficult due to thelatent character of the virus which can remain inactive within theorganism for a long time before actually manifesting itself,particularly as a result of stress.

Consequently, the conception of a process allowing lines of transgenicpigs to be produced constitutively resistant to the PRV virus or evenlines of transgenic cows constitutively resistant to the BHV-1 viruswould be of considerable economic interest.

The object of the invention is to propose such a process.

This has been possible due to previous research carried out on a mousemodel which, just like the pig or the cow, is sensitive to certainalphaherpesviruses and in particular to the HSV1 and PRV viruses.

It is known that the alphaherpesviruses are linked to the cells first ofall thanks to the interaction of a viral glycoprotein gC, entering theconstitution of the virion and heparan sulphate membrane present on thesurface of the cells, whilst subsequent fusion between the virionenvelope and the cell membrane then produces other glycoproteins (gB,gD, gH and gL).

Numerous works have focussed on the study of potential receptors of thealphaherpesviruses present on the surface of the cells of host mammalshaving a fixing ability of the viral particle and possibly being able tothus neutralise its infectious power.

Four receptor proteins of the alphaherpesviruses have been identified todate, namely:

-   -   HVEM or HveA which is an entry mediator for the HSV1 and HSV2        viruses but not the PRV virus (Montgomery, R. I., Warner, M. S.,        Lum B. J., and Spear, P. G. (1996). Herpes simplex virus-1 entry        into cells mediated by a novel member of the TNF/NGF receptor        family. Cell 87, 427-436).    -   three members of the immunoglobulin superfamily (HveB or        nectin-2; HveC or nectin-1 and HveD)

(Cocchi, F., Menotti, L., Mirandola, P., Lopez, M., andCampadelli-Fiume, G. (1998). The ectodomain of a novel member of theimmunoglobulin subfamily related to the poliovirus receptor has theattribute of a bona fide receptor for herpes simplex virus types 1 and 2in human cells. J. Virol. 72, 9992-10002;

Geraghty, R. J., Krummenacher, C., Eisenberg, R. J., Cohen G. H., andSpear, P. G. (1998) Entry of alphaherpesviruses mediated by poliovirusreceptor related protein 1 and poliovirus receptor. Science 280,1618-1620;

Shukla, D., Liu, J., Blaiklock, P., Shworak, N. W., Bai, X., Esko, J.D., Cohen, G. H., Eisenberg, R. J., Rosenberg, R. D., and Spear, P. G.(1999). A novel role for 3-O-sulfate heparan sulfate in herpes simplexvirus entry. Cell 99, 13-22;

Warner, M. S., Martinez, W., Geraghty, R. J., Montgomery, R. I.,Witbeck, J. C., Xu, R., Eisenberg, R. J., Cohen, G. H., and Spear, P. G.(1998). A cell surface protein with herpesvirus entry activity (HveB)confers susceptibility to infection by herpes simplex virus type 2,mutants of herpes simplex virus type 1 and pseudorabies virus. Virology246, 179-189.

According to the publications Spear, P. G. (1993). Entry ofalpha-herpesviruses into cells. Semin. Virol. 4, 167-180;Campadelli-Fiume, G., Arsenakis, M., Farabegoli, F., and Roizman, B.(1988). Entry of herpes simplex virus 1 in BJ cells that constitutivelyexpress viral glycoprotein D is by endocytosis and results indegradation of the virus. J. Virol. 62, 159-167, it has been suggestedthat in addition to the initial link with the heparan sulphate, it isthe interaction of the glycoprotein gD of the alphaherpesvirus with areceptor present on the surface of the cell which allows entry of thevirus in infectious form and that in certain types of cells, the HSV-1,PRV and BHV-1 viruses can use a common receptor of the glycoprotein gDto enter the cell.

It has therefore been proved that the protein HVEM can allow entry ofthe HSV1 and HSV2 viruses in the non-permissive cells, but not that ofthe PRV virus.

On the other hand, it has been demonstrated that the protein HveC, andparticularly that of the pig, behaves like a functional receptor notonly of the HSV. 1 virus but also of the animal alphaherpesviruses PRVand BHV-1 (Milne, R. S. B., Connolly, S. A., Krummenacher, C.,Eisenberg, R. J., and Cohen, G. H. (2001). Porcine HveC, a member of thehighly conserved HveC/nectin 1 family, is a functional alphaherpesvirusreceptor. Virology 281, 315-328; Cocchi, F., Menotti, L., Mirandola, P.,Lopez, M., and Campadelli-Fiume, G. (1998). The ectodomain of a novelmember of the immunoglobulin subfamily related to the poliovirusreceptor has the attribute of a bona fide receptor for herpes simplexvirus types 1 and 2 in human cells. J. Virol. 72, 9992-10002; Geraghty,R. J., Krummenacher, C., Eisenberg, R. J., Cohen G. H., and Spear, P. G.(1998) Entry of alphaherpesviruses mediated by poliovirus receptorrelated protein 1 and poliovirus receptor. Science 280, 1618-1620).

The abilities to bind to the viral glycoprotein gD are more particularlyattributed to the V domain for HveC and to the first two cystein-richdomains (“CRD”) for HVEM (Structure-Based Analysis of the Herpes SimplexVirus Glycoprotein D Binding Site Present on Herpesvirus Entry MediatorHevA (HVEM). Connolly S A, Landsburg D J, Carfi A, Wiley D C, EisenbergR J, Cohen G H; J virol 2002, Nov. 1; 76 (21): 10894-10904).

However, it has been shown in the publication by Martinez W M, Spear PG, Amino acid substitutions in the V domain of nectin-1 (HveC) thatimpair entry activity for herpes simplex virus types 1 and 2 but not forPseudorabies virus or bovine herpesvirus 1, J Virol. 2002 July; 76 (14):7255-62, that the abilities to bind with the viral glycoprotein gD ofthe protein HveC could be significantly changed without eliminating theabilities of this protein to trigger fusion of the cellular and viralmembranes and thus allow entry of the PRV and BHV-1 viruses in the cellin an infectious form.

It has also been shown in the publication by Campadelli-Fiume G, CocchiF, Menotti L, Lopez M. the novel receptors that mediate the entry ofherpes simplex viruses and animal alphaherpesviruses into cells Rev MedVirol. 2000 September-October; 10 (5): 305-19, that the protein HveCexpressed by the mouse can play a mediator role in the entry of thealphaherpesviruses PRV, BHV-1 and HSV independently of a detectableinteraction with the glycoprotein gD.

It has moreover been globally concluded in the study by Geraghty R J,Fridberg A, Krummenacher C, Cohen G H, Eisenberg R J, Spear P G, use ofchimeric nectin-1 (HveC)-related receptors to demonstrate that abilityto bind alphaherpesvirus gD is not necessarily sufficient for viralentry, Virology. 2001 Jul. 5; 285 (2): 366-75, that the interaction ofthe protein HveC with the glycoprotein gD was not sufficient to allowentry of the virus in the cell and that the characteristics of theprotein HveC responsible for the mediator properties for entry of theHSV, PVR and BHV1 viruses in the target cells could not be reduced toits abilities to bind with the glycoprotein gD.

It should furthermore be noted that the protein HveC has a remarkablywell conserved polypeptide sequence between the mammal species; by wayof example, 97% of the amino acids are common to the protein HveCexpressed by the pig and the protein HveC expressed in cows, whichimplies a strong identity of structure and function in these twospecies.

According to the publication by INOBE MANABU et al “Functional analysisof HVEM, a member of TNFR family, by using a transgenic mice expressingsoluble form of HVEM” (Biosciences information Service, Philadelphia,Pa.-US-2001.03.07), lines of genetically modified mice have been createdfor experimental purposes by introducing in the genome of these mice acoding transgene for a chimeric protein composed of the extracellulardomain of the protein HVEM and the crystallisable portion Fc of theimmunoglobulin IgG, in order to study the implication of the proteinHVEM in the regulation of the immune system.

It has thus been confirmed that the expression of a soluble form ofHveM, a member of the family of the receptor of TNFalpha, produces animmunosuppressor effect.

From this previous knowledge, according to the invention a process forproducing a mammal belonging to a non-human species rendered resistantby germinal transgenesis to infection by an alphaherpesvirus for whichthe polypeptide HveC or nectin 1 constitutes a functional receptor, isproposed, characterised in that a transgene allowing the expression of achimeric protein composed on the one hand of the extracellular domain ofnectin-1 or HveC or one of its parts and on the other the crystallisablefragment of an immunoglobulin, in particular a gamma typeimmunoglobulin, is introduced by insertion or homologous recombinationin the genome of the cells comprising the germinal line of the mammal orone of these ancestors, in an appropriate expression system.

The idea on which the invention is based therefore consisted of using inpart the mediator abilities of the protein HveC in relation to the entryof the targeted virus, but in a harmless way for the cell (by isolatingits cellular domain or one of its parts) so as to ultimately inhibit theentry of this virus into the cell and favour its elimination, by aprocess which is still to be determined.

The action mechanism of the chimeric protein expressed could inparticular comprise, thanks to the fixing ability of this both for theviral particle and the cellular receptor Fc, an increase in thephagocytosis and destruction abilities of the virions by the macrophagesand dendritic cells and an activation of the NK cytotoxic lymphocytes.

It could also comprise the formation of membrane receptors of thealphaherpesviruses in the form of functionally modified multimers byincorporating one or several units of the chimeric protein, allowing theviral particles to be fixed to the surface of the cell but not allowingthem to enter the cytoplasm in an infectious form.

According to the invention, the protein HveC or nectin-1 and/or theimmunoglobulin belong preferably to the homologous species.

Also only part of the nectin-1 or HveC could be used, or even, ifnecessary, the mutated forms of these parts, selected for their abilityto bind with the targeted virus.

The first stage of the process according to the invention thereforecorresponds to the preparation of the transgene which can be carried outusing methods well-known by people skilled in the art and abundantlydescribed in the literature consisting of cloning:

On the one hand, either complementary DNA of the RNA transcribed for thecellular receptor gene from an RNA preparation extracted from a tissuesample taken on a mammal, or the chromosome region (all exons andintrons) comprising the gene of this receptor from a genome DNApreparation also extracted from a tissue sample taken from a mammal, ora chimeric construction constituted for part of the cDNA and for theremaining part of the polypeptide chain of the corresponding genomefragment (“mini-gene”).

In all cases, only the part corresponding to the extracellular domain ofthis receptor will be used, or in another version of the process, asub-part of this domain or even, where necessary, a polypeptide sequenceessentially derived from this extracellular domain.

On the other hand, either the complementary cDNA of the RNA transcribedfor one of the heavy chain genes for the class and sub-class ofimmunoglobulin selected (for example G1) from an RNA preparationextracted from a tissue sample taken from a mammal, or the chromosomeregion (all exons and introns) comprising this heavy chain gene from agenome DNA preparation also extracted from a tissue sample taken from amammal, or a chimeric construction composed for part of the cDNA and forthe remaining part of the polypeptide chain of the corresponding genomefragment (“mini-gene”). The construction undertaken will advantageouslyretain the coding regions for the Hinge, CH₂ and CH₃ domains only of theheavy immunoglobulin chain selected (crystallisable fraction).

An example of such a construction for the human immunoglobulin G1 isdescribed in the publication CTLA-4 Is a Second Receptor for the B CellActivation Antigen B7. By Peter S. Linsley, William Brady, Mark Urnes,Laura S. Grosmaire, Nitin K. Damle, and Jeffrey A. Ledbetter; J. Exp.Med. © The Rockefeller University Press; volume 174, September 1991,561-569.

The cloning operations will be carried out from existing previousknowledge relating to the genes used, that is their sequence, theirchromosome localisation, if possible in the homologous species, butbeing based on the known sequences for this gene in other mammals.

These operations could be carried out by a polymerisation chain reaction(PCR) preceded by a reverse transcription stage for the complement DNAor by detection within banks of genome DNA of the targeted species ofthe clones likely to be hybridised with a specific probe or to produce aspecific PCR amplicon of the gene researched.

This construction will be carried out so as to join the coding sequencesfor the extracellular domain of nectin-1 or HveC or one of its parts at5′ of the coding sequences for the crystallisable fragment of the heavyimmunoglobulin chain (terminated by a stop codon) whilst complying withthe original reading framework of the two genes, and possibly the natureand effectiveness of the intron-extron junctions if they have beenincluded, so as to ultimately ensure the expression of a chimericprotein constituted for its terminal amino part of the polypeptidecorresponding to the extracellular domain of the HveC cellular receptoror one of its sub-parts and for its terminal carboxy part of the Hinge,CH2 and CH3 domains of the heavy immunoglobulin chain.

This construction will be undertaken in an expression vector allowing astrong expression of the chimeric protein in one or several biologicalcompartments of the host where it will allow protection of the cells soas to render the host globally resistant to the initial infection or toits development. The process will consist in particular of using activeexpression systems either constitutively in all the cells or morespecifically in the target tissues of the viral infection such as thetissues of the central nervous system or the epithelial tissues (inparticular those of the respiratory system).

The expression vector could comprise a promoter region, a terminationsignal, stimulator elements of the transcription, isolator sequences ofthe chromatin context, other transcription units and all elements likelyto ensure the desired expression.

The process will consist advantageously of using expression systemsconstituted from cloned regulator sequences in the homologous species,or for application to animals for production, in other animals normallyreared for human consumption.

The second stage of the process according to the invention consists ofintroducing the transgene thus obtained in the genome of the cellscomprising the germinal line of the targeted host mammal by insertion orhomologous recombination, again by a method well-known to personsskilled in the art such that this transgene is integrated in the geneticinheritance of this mammal.

Pronuclear micro injection of the DNA segment encoding the transgene ornuclear transfer of cells transformed in culture by the transgene inparticular can be used.

The invention also relates to a mammal belonging to a non-human speciesrendered resistant by germinal transgenesis to an infection by analphaherpesvirus for which the polypeptide HveC or nectin-1 constitutesa functional receptor by the effect of the expression of a chimericprotein composed on the one hand of the extracellular domain of thenectin-1 or HveC or one of its parts preferably of the homologousspecies, and on the other of the crystallisable fragment of animmunoglobulin, particularly of a gamma type immunoglobulin preferablyof the homologous species.

The invention also relates to the progeny of such a mammal, havinginherited by descent the transgene inserted in the genome of thegerminal line of one of its parents.

According to the invention, the alphaherpesvirus can advantageously bethe PRV virus and the mammal belong to the porcine species.

According to a variation of the invention, the alphaherpesvirus can alsobe the BHV-1 virus and the mammal belong to the bovine species.

It is essential according to the invention that the transgenesisoperation is a germinal transgenesis such that the progeny of the mammalare also likely to express the chimeric protein.

The invention also relates to a genetic material such as the semen orooecytes or embryos essentially derived from transgenic mammals of theabove-mentioned type.

Several examples of sequences of chimeric proteins according to theinvention are attached.

These are sequences of amino acids which comprise the signal peptide atthe terminal amino end which will be processed during maturation.

A chimeric protein according to this model is also described in thedocument JP-2001-328430 within the framework of another application.

The feasibility of the process according to the invention has beenconfirmed by

1) experimental results obtained within the framework of the in vivoanalysis of the resistance to the human herpes simplex virus type 1(HSV-1) of transgenic mice expressing a chimeric protein Hvem—Ig.

According to these tests, a coding transgene for a chimeric proteincomposed of the extracellular domain of the murine receptor HVEM of thisvirus and the crystallisable portion Fc of the human immunoglobulinIgG-1 was introduced by germinal transgenesis into the DNA of thesemice.

The extracellular murine HveM domain was cloned by RT PCR from apreparation of RNA extracted from female rat cells stimulated byconcavaline A, obtained on stock mice BALB/c.

The primers used for the RT PCR reaction were5′-TAACTCGAGCTCTTGGCCTGAAGTTTC-3′ and5′-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3′.

The cDNA was inserted in the Xhol and BamHl restriction sites of aplasmid having the sequence of the crystallisable fragment of the humanimmunoglobulin G1 (as described in the publication by Nakagawa I.,Murakami, M., Ijima, K., Chikuma, S., Saito, I., Kanegae, Y., Ishikura,H., Yoshiki, T., Okamoto, H., Kitabatake, A., and Uede, T. (1998).Persistent and secondary adenovirus-mediated hepatic gene expressionusing adenovirus vector containing CTLA41gG. Human Gene Therapy 9,1739-1745).

The XhoI/XbaI fragment containing the DNA encoding the chimeric proteinHveMIg was isolated from this construction and inserted in turn, afterblunt-ending, in the SwaI restriction site of the cosmid vector pAxCAwt(commercially distributed by the Company TAKARA, Kyoto, Japan) under thecontrol of the CAG promoter (β actin promoter) known to allow a highconstitutive expression in any type of cell (Niwa, H., Yamamura, K., andMiyazaki, J. (1991). Efficient selection for high-expressiontransfectants with a novel eukaryotic vector. Gene 108, 193-200).

Three lines A, B, C of transgenic mice each from an independent founderexpressing this chimeric protein HVEM Ig, were then created bymicroinjection of the Pmel/Sall fragment of this vector containing thestimulation factor of the viral gene CMV IE transcription, the promoterof the Beta actin gene of the chicken, the sequence of the proteinHVEMIg and the polyadenylation 3′ signal of the Beta globin locus of therabbit, in the fertilised embryo pronuclei of mice (genotype F1 C57BL/6×SJL).

The presence of the protein HVEMIg was detected as a specific bandrevealed by an anti-HVEMIg antibody (produced by hyperimmunisation onthe rabbit) by immunoelectrophoresis in the serum of the three lines oftransgenic mice, with a lower average content for the mice of line B.

The construction carried out is shown schematically in FIG. 1.

The concentration of chimeric protein HVEMIg in the serum of the mice ofthe three transgenic lines A B C is shown in Tables 1, 2, 3 and 4attached.

The transgenic mice of three lines developed normally and no differenceswere noted between the weights of these mice and those of theirnon-transgenic homologs from the same litter.

To determine whether the transgenic mice expressing the chimeric proteinHVEMIg were effectively protected against the HSV-1 virus, transgenicmice and non-transgenic control mice from the same litter wereinoculated intravenously and a dose of 10⁹ cfu corresponding to tentimes the lethal dose (10 LD 50) of virus.

The LD 50 was determined initially in the less sensitive of the twolines of mice used to produce the hybrid transgenic animals.

According to FIGS. 2, 3 and 4, the transgenic T_(g) mice respectively oflines A B and C and the non-transgenic T_(g) mice still alive up to 14days after infection were counted.

It could also be noted that all the lines of transgenic mice wereresistant to the HSV-1 virus.

More specifically, all the transgenic mice of lines A and C survivedinoculation with the virus and remained in good health for severalmonths after the trial (Tables 1 and 4).

Only one transgenic mouse of line B died after inoculation with thevirus whilst the other six survived (Table 3), but line B is the one forwhich the serum rates measured for HVEMIg were the lowest.

On the other hand, six of the seven non-transgenic mice from the samelitters as the transgenic mice of line A, 13 of the 14 non-transgenicmice from the same litters as the transgenic mice of line B and six ofthe seven non-transgenic mice from the same litters as the transgenicmice of line C developed symptoms such as paralysis or died in the 14days following inoculation with the HSV-1 virus.

The expression of the LAT of the HSV-1 virus in the trigeminal gangliaof the surviving mice after inoculation by the method described in thepublications was investigated.

-   -   Spivak, J. G., and Fraser, N. W. (1987). Detection of herpes        virus type 1 transcripts during latent infections in mice. J.        Virol., 61, 3841-3847.    -   Stevens, J. G., and Cook, M. L. (1971). Latent herpes simplex        virus in spinal ganglia of mice. Science 173, 843-845.

The RT-PCR method was thus used to detect the LAT expression.

This was observed in the non-transgenic mice which developed symptomsand in a single transgenic mouse of line B which did not presentsymptoms, but was not on the other hand observed in any of the othertransgenic mice tested nor in the surviving non-transgenic mice whichdid not develop symptoms.

Within the framework of this study, a control trial was also carried outin order to determine whether the transgenic mice expressing thechimeric protein HVEMIg were protected against the PRV virus when theprotein HVEM is not a functional receptor for the PRV virus.

Transgenic mice of line A and non-transgenic mice from the same litterwere for that purpose inoculated intravenously with a dose correspondingto 10 times the lethal dose (10 LD 50) of PRV virus (Table 2 and FIG.5).

It could also be noted that with the exception of a transgenic mousethat survived for 10 days, all the mice died in the five days followinginoculation with the PRV virus.

Subsequently, the transgenic mice expressing the chimeric protein HVEMIgdid not prove to be protected against the PRV virus.

Within the framework of this study, it was also sought to determinewhether the resistance of the transgenic mice to inoculation with theHSV-1 virus which could be noted in vivo accompanied in parallel aresistance of the cells of these mice once isolated.

Cultures of embryonic fibroblasts of transgenic mice or non-transgenicmice were therefore inoculated with the HSV-1 virus.

The lysis ranges caused by the virus in the cellular culture 5 daysafter inoculation were then counted and it was noted that the number ofthese ranges was clearly higher in the case of the fibroblasts ofnon-transgenic mice than in the case of fibroblasts of transgenic mice(on average 22 plaques per culture disk for the non-transgenic mice and1 plaque per culture disk for the transgenic mice).

A similar test for the PRV virus was carried out in parallel byinoculating cultures of embryonic fibroblasts from transgenic mice ornon-transgenic mice with the PRV virus without noting any significantdifferences between the number of lysis ranges observed in thetransgenic mice and in the non-transgenic mice.

These results are likely to prove that the chimeric protein HVEMIgexpressed by the fibroblasts of the transgenic mice is involved in theinhibition of the adsorption of the HSV-1 virus by the embryonicfibroblasts.

In a complementary test, the results of which are shown in Table 5, itwas investigated whether the chimeric protein HVEMIg present in theserum of transgenic mice could inhibit the infection of cellularcultures by the HSV-1 or PRV viruses.

Serum was therefore collected from transgenic mice of line C and theHSV-1 virus or PRV virus incubated with an inoculate of this serum priorto bringing it into contact with the Vero cell cultures.

It could thus be established that the serum of transgenic mice of line Ccan protect Vero cells from contamination by the HSV-1 virus but notfrom contamination by the PRV virus.

A control test carried out with serum from non-transgenic mice was incontrast not able to note any antiviral activity.

It was moreover noted that the serum from transgenic mice of line C nolonger presents any antiviral activity after it was brought into contactwith a polyclonal anti-HVEMIg serum produced by hyperimmunisation on therabbit for 30 minutes at ambient temperature.

This last result confirms that the seroneutralising abilities of theserum of the transgenic mice can be attributed to the expression of thechimeric protein.

All of these results demonstrate that the antiviral activity noted comesfrom the chimeric protein HVEMIg present in the serum of the transgenicmice, but are also without doubt associated with the expression of thischimeric protein on the surface of the cells of the host just as theembryonic fibroblasts evaluated in these tests.

These results confirm the antiviral effectiveness of the in-vivoexpression of a modified form of a membrane receptor of thealphaherpesviruses, despite the immunosuppressive properties describedin the case of the protein HveM.

2) experimental results obtained within the framework of the in vivoanalysis of the resistance to the PRV virus of transgenic miceexpressing a chimeric protein Hvec-Ig.

According to these tests, a coding transgene for a chimeric proteincomposed of the extracellular domain of the porcine receptor HveC of thePRV virus and the crystallisable portion Fc of the human immunoglobulinIgG-1 was introduced by germinal transgenesis into the DNA of thesemice.

The porcine HveC extracellular domain was cloned by RT PCR from an RNApreparation extracted from pig cells.

The primers used for the RT PCR reaction were5′-TAACTCGAGCTCTTGGCCTGAAGTTTC-3′ and 5′-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3′as described and following the conditions proposed in the publication(Milne, R. S. B., Connolly, S. A., Krummenacher, C., Eisenberg, R. J.,and Cohen, G. H. (2001). Porcine HveC, a member of the highly conservedHveC/nectin 1 family, is a functional alphaherpesvirus receptor.Virology 281, 315-32).

The cDNA was inserted into the Xhol and BamHl restriction sites of aplasmid having the sequence of the crystallisable fragment of the humanimmunoglobulin G1 (as described in the publication by Nakagawa I.,Murakami, M., Ijima, K., Chikuma, S., Saito, I., Kanegae, Y., Ishikura,H., Yoshiki, T., Okamoto, H., Kitabatake, A., and Uede, T. (1998).Persistent and secondary adenovirus-mediated hepatic gene expressionusing adenovirus vector containing CTLA41gG. Human Gene Therapy 9,1739-1745). The XbaI/XbaI fragment of this plasmid containing the DNAencoding the HveC-Ig fusion was isolated, blunt-ended and bound to theSalI adaptors, in order to insert it in turn, after digestion by Xho1and Sal1, in the XhoI restriction site of the vector pCXN2 under thecontrol of the CAG promoter (β actin promoter) known to allow a highconstitutive expression in any type of cell (Niwa, H., Yamamura, K., andMiyazaki, J. (1991). Efficient selection for high-expressiontransfectants with a novel eukaryotic vector. Gene 108, 193-200). Therecombinant plasmid thus obtained was designated Pcxn2/pHvecIg.

Six lines #6, #22, #32, #33, #37 and #45 of transgenic mice each from anindependent founder expressing this chimeric protein HveC-Ig werecreated by microinjection of the SalI/SalI fragment of this plasmidcontaining the stimulation factor of the transcription of the viral geneCMV IE, the promoter of the Beta actin gene of the chicken, the sequenceof the protein HVEMIg and the polyadenylation 3′ signal of the Betaglobin locus of the rabbit, in fertilised embryo pronuclei of mice(strain C57/BL6).

The construction carried out is shown schematically in FIG. 6.

The transgenic mice of the six lines developed normally and nodifferences were noted between the weights of these transgenic mice andthe weight of the non-transgenic mice from the same litter.

Viral Trials by Intra-Peritoneal Injections

To determine whether the transgenic mice expressing the chimeric proteinHveC-Ig were effectively protected against the PRV virus, a dose of 500p.f.u. corresponding to twenty times the lethal dose (20 LD 50) of viruswas inoculated in a first trial series intraperitoneally in transgenicmice and non-transgenic control mice from the same litter.

According to Table 6, the transgenic T_(g) mice respectively from lines#6, #22, #32, #33, #37 and #45 and the non-transgenic T_(g) mice stillalive up to 14 days after infection were counted.

It could thus be noted that the 6 lines of transgenic mice wereresistant to the PRV virus in this renowned strict trial, with 100%survival of the transgenic animals with the exception of a single animalfor line #33.

On the other hand, only around 9% of the non-transgenic mice survived,with a variation according to the trials conducted for each line.

These trials were confirmed for lines #22 and #32 in an independentlaboratory using a different strain of the PRV virus.

Viral Trials by Intranasal Infection

In a second series of trials, transgenic and non-transgenic control micefrom the same litter were inoculated intranasally with a dose of 250p.f.u. corresponding to ten times the lethal dose (20 LD 50) of virus.

According to Table 7, the transgenic T_(g) mice respectively from lines#6, #22, #32, #33 and #37 and the non-transgenic non T_(g) mice stillalive up to 14 days after infection were counted.

It could thus be noted that the 5 lines of transgenic mice wererelatively resistant to the PRV virus in this renowned strict trial,with around 70% survival of the transgenic animals.

On the other hand, only around 10% of the non-transgenic mice survived,with a variation according to the trials conducted for each line.

These results demonstrate an effective protective action of thetransgene allowing the expression of the chimeric protein HveC-Ig in thecontext of a strict trial in the mouse by intranasal administration.

Overall, all these results show the effectiveness in-vivo of theprotection conferred to a mammal by the process according to theinvention, based on the inhibition of the entry of an alphaherpesvirusin the target cell. This effectiveness is only possible because theprocess proposed probably allows several levels of action, themechanisms of which are not all fully explained: in addition to thebinding abilities of the viral proteins gD known for the HveM and HveCreceptors, the use of the extracellular domain alone allows its receptorabilities to be isolated from the complex biological functions of thesetrans-membrane proteins in their entry and in particular their aptitudeto initiate intracellular signalling cascades.

This allows a priori envisaging of the superexpression of this proteindomain without nevertheless amplifying the physiological function andwithout doubt allows the absence of secondary effects observed for thetransgenic animals expressing the chimeric protein.

This probably allows in the context of an infection in vivo:

-   -   competition to fix the viral particles infecting chimeric        proteins in solution or present on the surface of the cells with        the functional membrane receptors of the virus;    -   neutralisation of the viral particles by opsonisation and        increasing of the phagocytosis by the macrophages and dendritic        cells;    -   activation of the cellular immunity by stimulation of the NK        lymphocytes.

The effectiveness of the process in the case of experimental infectionsby intranasal administration together with (for HveMIg) the resistanceof embryonic fibroblasts from transgenic animals move towards effectiveinhibition of the entry of the virus in the sensitive target cells bythe membrane fraction of the chimeric protein expressed, beyond theseroneutralising ability of the soluble fraction secreted in the serum.

This inhibition of the entry of the virus in the cell could be theresult of modified membrane receptors of the virus according to adominant method, authorising fixing of the virus to the surface of thetarget cells but not its entry in the cytoplasm in an infectious form.

The effectiveness of the process on the entry of the virus and the firstresults relating to the viral latency after infection (in the case ofHveM and HSV1) moreover allow the absence of latency in the breedinganimals thus protected to be envisaged, which is not necessarily thecase for vaccination strategies, and therefore a more effective controlof any resurgence of the infection.

3) Experimental results obtained within the framework of the in vitroanalysis of the resistance to the BHV-1 and PRV virus of cellular linestransformed by plasmids expressing chimeric proteins constructed fromthe extracellular domain of the porcine protein HveC and thecrystallisable fragment of the human immunoglobulin Ig.

According to this test, lines of Vero cells transformed by plasmidsexpressing a soluble form of the porcine protein HveC inhibiting entryof the BHV-1 and PRV viruses were prepared and then the resistance ofthese cellular lines to infection by these viruses was analysed todetermine the antiviral properties of the soluble form of the porcineprotein HveC in vitro.

To construct a plasmid expressing a soluble form of the porcine proteinHveC (PHveCIg) an expression vector PCXN2 was used allowingtranscription of a coding messenger RNA for the extracellular domain ofthe porcine protein HveC together with the crystallisable fragment Fc ofthe human immunoglobulin IgG1.

The extracellular porcine domain HveC was cloned by RTPCR from an RNApreparation extracted from pig cells.

The primers used for the RT PCR reaction were5′-TAACTCGAGCTCTTGGCCTGAAGTTTC-3′ and 5′-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3′as described and according to the conditions proposed in the publication(Milne, R. S. B., Connolly, S. A., Krummenacher, C., Eisenberg, R. J.,and Cohen, G. H. (2001). Porcine HveC, a member of the highly conservedHveC/nectin 1 family, is a functional alphaherpesvirus receptor.Virology 281, 315-32).

The cDNA was inserted in the Xhol and BamHl restriction sites of aplasmid having the sequence of the crystallisable fragment of the humanimmunoglobulin G1 (as described in the publication by Nakagawa I.,Murakami, M., Ijima, K., Chikuma, S., Saito, I., Kanegae, Y., Ishikura,H., Yoshiki, T., Okamoto, H., Kitabatake, A., and Uede, T. (1998).Persistent and secondary adenovirus-mediated hepatic gene expressionusing adenovirus vector containing CTLA41gG. Human Gene Therapy 9,1739-1745). The XbaI/XbaI fragment of this plasmid containing the DNAencoding the HveC-Ig fusion was isolated, blunt-ended and bound to theSalI adaptors, in order to insert in turn in the XhoI restriction siteof the vector pCXN2 under the control of the known CAG promoter (β actinpromoter) to allow a high constitutive expression in any type of cell(Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selectionfor high-expression transfectants with a novel eukaryotic vector. Gene108, 193-200). The recombinant plasmid thus obtained was designatedPcxn2/pHvecIg.

Cellular lines transformed in a stable way were prepared by transfectionwith the plasmid thus obtained.

The cellular lines thus transformed were cultivated under conditionsallowing accumulation of the chimeric protein in the medium, that is 24hours of additional cultures after spreading at subconfluence.

These cultures were then inoculated with the PRV or BHV-1 viruses with amultiplicity of 50 p.f.u. per box of 35 mm diameter with an incubationtime of one hour followed by two rinses with DMEM prior to covering withthe medium DMEM 0.5% agar.

The lysis ranges caused by the virus in the cellular culture 4 daysafter inoculation were then counted. It was noted that in the cellularlines expressing the chimeric protein PHveCIg the number of lysisplaques was notably reduced by comparison with the control resistantcellular lines and the Vero lines four days after inoculation.

The results obtained are shown in Table 8.

The cellular lines transformed by the plasmid PCXN2/pHveC Ig are clearlyresistant to attack by the PRV and BHV-1 viruses.

4) Experimental results obtained within the framework of the in vitroanalysis of the resistance to the BHV-1 and PRV viruses of cellularlines transformed by plasmids expressing chimeric proteins constructedfrom the extracellular domain of the porcine protein HveC and thecrystallisable fragment of the pig immunoglobulin g1.

According to this test, lines of Vero cells transformed by plasmidsexpressing a soluble form of the porcine protein HveC inhibiting entryof the BHV-1 and PRV viruses were prepared, then the resistance of thesecellular lines to infection by these viruses analysed to determine theantiviral properties of the soluble form of the porcine protein HveC invitro.

To construct a plasmid expressing a soluble form of the porcine proteinHveC (PHveCIg) a pCXN2 expression vector was used allowing transcriptionof a coding messenger RNA for the extracellular domain of the porcineprotein HveC and the crystallisable fragment Fc of the porcineimmunoglobulin IgG-1.

The porcine HveC extracellular domain was cloned by RT PCR from an RNApreparation extracted from pig cells.

The primers used for the RT PCR reaction were5′-TAACTCGAGCTCTTGGCCTGAAGTTTC-3′ and 5′-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3′as described and according to the conditions proposed in the publication(Milne, R. S. B., Connolly, S. A., Krummenacher, C., Eisenberg, R. J.,and Cohen, G. H. (2001). Porcine HveC, a member of the highly conservedHveC/nectin 1 family, is a functional alphaherpesvirus receptor.Virology 281, 315-32).

The cDNA thus obtained was linked at 5′ of a coding fragment of cDNA forthe crystallisable Fc fragment of the porcine immunoglobulin Igcomplying with the original reading framework of the two polypeptides.

The cDNA fragment of porcine immunoglobulin was cloned from an RNApreparation extracted from lymphoid pig tissues from a line of the LargeWhite type (FHO25) by RT PCR using as a triggerTAACTCGAGCTCTTGGCCTGAAGTTTC-3′ and 5′-TTAAGGATCCGAGGAGCAGGTGGTGTCT-3′according to the conditions proposed in the publication by Simon MusyokaMwangi, Thomas J. Stabel*, Marcus E. Kehrli Jr, development of abaculovirus expression system for soluble porcine tumor necrosis factorreceptor type I and soluble porcine tumor necrosis factor receptor typeI-IgG fusion protein, Veterinary Immunology and Immunopathology 86(2002) 251-254.

The cDNA resulting from the fusion codes for a chimeric protein, theamino acids sequence of which is attached (sequence 4).

The cDNA resulting from the fusion was inserted in the Xhol restrictionsite of the plasmid pCXN2 under the control of the promoter of the betaactin gene of the chicken associated with the stimulation factor of thetranscription of the viral gene CMV IE and the polyadenylation sequenceof the beta globin of the rabbit.

The resulting plasmid thus obtained was designated PCXN2/pVCC-pFc.

A restricted version of this plasmid designated PCXN2/pV-pFc wasconstructed using only the V domain of the protein HveC and thecrystallisable fragment Fc of the pig immunoglobulin IgG.

The amino acids sequence of the chimeric protein thus obtained isattached (sequence 3).

Cellular lines transformed in a stable manner were prepared bytransfection with the plasmid thus obtained.

The cellular lines thus transformed were cultivated under conditionsallowing accumulation of the chimeric protein in the medium, that is 24hours of additional cultures after spreading at subconfluence.

These cultures were then inoculated with the PRV and BHV-1 viruses witha multiplicity of 50 p.f.u. per box of 35 mm diameter with an incubationtime of one hour followed by two rinses with DMEM before covering withthe medium DMEM 0.5% agar.

The lysis ranges caused by the virus in the cellular culture three daysafter inoculation were counted and the findings shown in Table 9.

The cellular lines transformed by the two versions of the transgeneexpressing a chimeric protein according to the invention are clearlyresistant to attack by the BHV-1 and PRV viruses. TABLE 1 Resistance oftransgenic mice of line A to inoculation with the HSV-1 virus Number ofTest animal Sex HVEMIg (ug/ml) Symptoms Day of death  I A1 * M 14.8 −A2 * M 10.0 − A3 * M 11.6 − A4 * F 7.6 − A5 * F 8.3 − A6 * F 10.0 − A7 *F 24.6 − A8 * F 8.5 − A9 * F 7.9 − A10 * F 7.3 − L1 M 1.5 − L2 M 1.1 −L3 M 1.6 + 4 L4 M 0.8 + 14 L5 M 1.6 + L6 F 0.6 + 5 II A11 * M 10.1 −A12 * M 15.9 − A13 * M 10.5 − A14 * M 9.3 − A15 * F 7.3 − A16 * F 14.0 −A17 * F 15.4 − A18 * F 14.9 − L7 M 0.4 + 4 L8 M 0.5 + L9 F 0.5 +

TABLE 2 Sensitivity of transgenic mice of line A to inoculation by thePRV virus Number of Day of animal Sex HVEMIg (ug/ml) Symptoms deathA19 * M 41.2 + 4 A20 * F 19.4 + 10  A21 * F 29.6 + 5 A22 * F 24.2 + 5A23 * F 20.2 + 5 C57BL/6 M NT + 4 C57BL/6 M NT + 4 C57BL/6 M NT + 5C57BL/6 M NT + 5 C57BL/6 M NT + 5

TABLE 3 Resistance of transgenic mice of line B to inoculation by theHSV-1 virus Number of HVEMIg Day of Test animal Sex (ug/ml) Symptomsdeath RT-PCR  I B1 * M 8.5 − − B2 * M 7.8 − − B3 * M 7.7 − − B4 * F8.1 + 11 L1 M 0.4 + 6 L2 M 0.7 + 6 L3 M 0.5 + 5 L4 M 0.7 + 7 L5 F 0.3 +5 II B5 * M 5.0 − + B6 * M 6.2 − − B7 * F 4.4 − − L6 M 0.6 − L7 M 0.6 +10 L8 M 0.5 + 7 L9 M 0.6 + + L10 F 0.4 + 7 L11 F 0.5 + 5 L12 F 0.5 + 5L13 F 0.5 + 5 L14 F 0.4 + 6

TABLE 4 Resistance of transgenic mice of line C to inoculation by theHSV-1 virus Number of HVEMIg Day of Test animal Sex (ug/ml) Symptomsdeath RT-PCR C1 * M 20.4 − − C2 * M 14.2 − − C3 * F 19.3 − − C4 * F 18.5− − C5 * F 24.3 − − C6 * F 21.8 − − C7 * F 24.0 − − C8 * F 20.6 − − L1 M1.3 − − L2 M 0.9 + + L3 M 1.6 + 6 L4 F 1.3 + 5 L5 F 1.4 + 5 L6 F 1.5 + 6L7 F 1.6 + 5

In Tables 1 to 4, the animals marked with an * are transgenic animalswhilst the animals marked with an L are non-transgenic control animalsfrom the same litters. TABLE 5 Neutralisation of the HSV-1 virus by thechimeric protein HVEMIg in the serum of transgenic mice of line C.Inoculation of HSV-1 virus on VERO cells after incubation with variableconcentrations of this serum Number of lysis ranges observed Serum(HVEMIg ug/ml) HSV-1 PRV (20.4) 0 108.0 ± 8.8  (2.04) 0 —  (0.20) 1.7 ±1.6 —  (0.02) 34.7 ± 16.2 —  (0.20) + anti HVEMIg 30.3 ± 6.9  — Control44.0 ± 0   107.3 ± 2.9

TABLE 6 Trials by intraperitoneal administration (20 LD 50) Number ofNumber of % Number controls animals Number of % sur- of (of the Tgcontrols survival vival animals same surviving surviving trans- of Tglitters) at 14 at 14 genic con- Line tested tested days days animalstrols PhveCIg#6 5 10 5 2 100 20 PhveCIg#22 12 12 12 0 100 0 PhveCIg#3210 7 10 1 100 14.2 PhveCIg#33 7 9 6 0 85.7 0 PhveCIg#37 10 4 10 1 100 25PhveCIg#45 3 12 3 1 100 8.3

TABLE 7 Trials by intranasal administration (10 LD 50) Number of Numberof % Number controls animals Number of % sur- of (of the Tg controlssurvival vival animals same surviving surviving trans- of Tg litters) at14 at 14 genic con- Line tested tested days days animals trols PhveCIg#68 7 4 0 50 0 PhveCIg#22 23 21 18 2 78.3 9.5 PhveCIg#32 10 8 6 1 60 12.5PhveCIg#33 17 8 11 1 64.7 12.5 PhveCIg#37 10 7 9 1 90 14.2

TABLE 8 Resistance of transformed cellular lines to the PRV and BHV-1alphaherpesviruses Cellular Number of lysis ranges observed line*PHveCIg PRV BHV-1 A6 + 0 0 C1 + 0 0 C2 − 55.8 + 4.6 75.5 + 3.5 Vero −50.8 + 6.2 65.0 + 5.6* In this table, lines A6 and C1 are cellular lines transformed by theplasmid pCXN2/pHveCIg and expressing the chimeric protein PHveCIg whilstline C2 corresponds to a negative reference of this transformation notexpressing the transgene and the Vero line to the initial cellssensitive to the viruses and used for production of the linestransformed by the different transgenes.

TABLE 9 Resistance of the transformed cellular lines to the PRV andBHV-1 alphaherpesviruses Number of lysis ranges observed Cellular line*PRV BHV-1 3-16 0 1 V110 0 6 Vero 56  67 *In this table, line 3-16 is a cellular line transformed by the plasmidp CXN2/pVCC - pFc and line V110 is a cellular line transformed by theplasmid p CXN2/pV - pFc whilst the Vero line corresponds to the initialcells sensitive to the viruses and used for the production of cellstransformed by the different transgenes.

1. Process for producing a mammal belonging to a non-human speciesrendered resistant by germinal transgenesis to an infection by analphaherpesvirus, for which the polypeptide HveC or nectin-1 constitutesa functional receptor, characterized in that a transgene allowing theexpression of a chimeric protein composed on the one hand of theextracellular domain of the nectin-1 or HveC or of one of its parts andon the other of the crystallisable fragment of an immunoglobulin, isintroduced by insertion or homologous recombination in the genome of thecells constituting the germinal line of the mammal, in an appropriatesystem of expression.
 2. Process according to claim 1, characterized inthat the immunoglobulin is a gamma type immunoglobulin.
 3. Processaccording to claim 1, characterized in that the nectin-1 or HveC and/orimmunoglobulin belong to the homologous species.
 4. Mammal belonging toa non-human species, characterized in that it has been renderedresistant by germinal transgenesis to an infection by analphaherpesvirus for which the polypeptide HveC or nectin-1 constitutesa functional receptor by the effect of the expression of a chimericprotein composed on the one hand of the extracellular domain of thenectin-1 or HveC, preferably of the homologous species, and on the otherhand of the crystallisable fragment of an immunoglobulin, notably agamma type immunoglobulin, preferably of the homologous species. 5.Mammal according to claim 4, characterized in that the specific receptorof the alphaherpesvirus is a sub-part of the extracellular domain ofnectin-1 or HveC.
 6. Mammal according to claim 4, characterized in thatit belongs to the porcine species and the alphaherpesvirus is the PRVvirus.
 7. Mammal according to claim 4, characterized in that it belongsto the bovine species and the alphaherpesvirus is the BHV-1 virus. 8.Mammal according to claim 4, characterized in that it contains in thegenome of its cells a coding transgene for a chimeric protein composedon the one hand of the extracellular domain of nectin-1 or HveC or ofone of its parts, and on the other of the crystallisable fragment of animmunoglobulin, in an appropriate expression system, this transgenehaving been inserted in the genome of the germinal line of one of itsparents.
 9. Genetic material such as semen or ooecyte or embryoessentially derived from the mammal according to claim 4.